Modified meningococcal polysaccharide conjugate vaccines

ABSTRACT

The invention relates to chemically-modified group B polysaccharides of  Neisseria meningitidis . The invention also provides vaccines in which the respective modified polysaccharides are conjugated to a protein carrier, and the like. More specifically, the present invention provides novel group B meningococcal unsaturated N-acyl derivative polysaccharides, novel conjugates of the group B meningococcal unsaturated N-acyl derivative polysaccharides, pharmaceutical compositions comprising conjugate molecules of group B meningococcal unsaturated N-acyl derivative polysaccharide fragments covalently bound to proteins, and the use of these compositions as vaccines.

FIELD OF THE INVENTION

[0001] This invention relates to chemically-modified group Bpolysaccharides of Neisseria meningitidis. This invention also providesvaccines in which the respective modified polysaccharides are conjugatedto a protein carrier, and the like.

BACKGROUND OF THE INVENTION

[0002] Meningitis caused by group B N. meningitidis and E. coli K1remain major world health problems. Group B meningitis occurs in bothendemic and epidemic situations and accounts for approximately half ofall recorded cases of meningococcal meningitis, while K1-positive E.coli are the leading cause of meningitis in neonates. Currently there isno vaccine commercially available against disease caused by group Bmeningococci and E. coli K1. This is in large part due to the fact thatthe group B meningococcal polysaccharide (GBMP) is only poorlyimmunogenic in humans. This poor immunogenicity of native GBMP andresulting immune tolerance has been postulated to be due to the presenceof a common epitope in human and animal tissue. There are some recentlyreported candidate vaccines based on complexes of the GBMP with outermembrane proteins, but, as yet, there is no clear evidence of theirefficacy in humans.

[0003] Recently, a new concept of a vaccine based on a syntheticchemically modified (N-propionylated) group B polysaccharide-protein(N-Pr-GBMP-protein) conjugate has been developed. The vaccine induces inmice high titers of IgG antibodies which are not only protective, butalso cross-react with unmodified GBMP (i.e. N-acetyl-GBMP). This conceptis described and claimed in U.S. Pat. No. 4,727,136, issued Feb. 23,1988 to Harold J. Jennings, et al.

[0004] It has been inferred that a vaccine which raises cross-reactiveantibodies, such as that described in U.S. Pat. No. 4,727,136, couldonly be successful at the expense of breaking immune tolerance. Thishypothesis is legitimized by the identification of a common epitopeconsisting of a chain of α-(2-8)-linked sialic acid residues (with aminimum requirement of ten residues) in both the native N-Ac-GBMP and inhuman and animal tissue (Jennings, Contrib. Microbiol. Immunol. Basel,Karger, 1989, Vol. 10, 151-165). These polysialosyl chains function asdevelopmental antigens and have for the most part been associated withthe fetal state in embryonic neural cell adhesion (Finne et al, Biochem.Biophys. Res. Commun., 1983, 112, 482). During post-natal maturation,this antigen is down-regulated (Friedlander et al, J. Cell Biol. 1985,101, 412) but is expressed in mature humans during the regeneration ofdiseased muscles (Cashman et al, Ann. Neuron., 1987, 21, 481) in tumorcells (Roth et al, Proc. Natl. Acad. Sci., 1988, 85, 299) and in naturalkiller (NK) and CD3 ⁺T cells (Husmann et al, Eur. J. Immunol., 1989, 19,1761. Although the consequences of breaking tolerance to these fetalantigens have not yet been established, it is desirable to developvaccines which have reduced immunogenicity for human epitopes.

[0005] Therefore, an object of the present invention is to developmodified group B meningococcal polysaccharides which are immunogenic yetinduce antibodies which have reduced cross-reactivity with nativeepitopes of the host. It is another object to providepolysaccharide-protein conjugates which comprise these modifiedpolysaccharides. Another object of this invention is to provide vaccineshaving immunogenic properties which exhibits substantially reducedcross-reactivity with GBMP.

SUMMARY OF THE INVENTION

[0006] The present invention generally provides chemically-modifiedgroup B polysaccharides of Neisseria meninigtidis. The present inventionalso provides for vaccines in which the respective modifiedpolysaccharides are conjugated to a protein carrier.

[0007] Specifically, this invention provides for unsaturated group BN-acyl derivative polysaccharides of N. meningitidis, conjugates of theunsaturated N-acyl derivative polysaccharide covalently bound toproteins, pharmaceutical compositions comprising conjugate molecules ofN. meningitidis unsaturated N-acyl derivative polysaccharides, and theuse of these compositions as vaccines.

[0008] In one aspect of the invention, there is provided a modified Bpolysaccharide of N. meningitidis having sialic acid residue N-acetyl(C₂) groups replaced by an unsaturated C₃₋₄ acyl group.

[0009] In another aspect, there is provided an antigenic conjugatecomprising unsaturated C₂₋₄ N-acyl derivative polysaccharides conjugatedto an immunologically suitable protein, having enhanced immunogenicitycompared to native polysaccharides with reduced inducement ofcross-reactive antibodies.

[0010] In a further aspect, there is provided a vaccine comprising theunsaturated N-acyl derivative polysaccharide-protein conjugate inassociation with a suitable carrier or diluent. The vaccines of theinvention may also comprise a therapeutically effective amount of anadjuvant suitable for human use, for example aluminum phosphate,aluminum hydroxide or stearyl tyrosine.

[0011] In a yet further aspect, there is provided a method of immunizingmammals against N. meningitidis and E. coli K1 infections, which methodcomprises administering parenterally to mammals subject to suchinfections, including humans, an immunologically effective amount of thevaccine of the invention. The vaccine is typically administered in anamount of about 1 to 50 micrograms per kilogram body weight, for example5 to 25, micrograms per kilogram body weight.

[0012] In yet another aspect, the invention provides serum and a gammaglobulin fraction capable of protection against meningitis caused bygroup B N. meningitidis and E. coli K1. The fraction is produced byimmunizing a mammal with a vaccine of the invention and preferablyseparating the gamma globulin fraction from the immune serum. Thefraction is then administered to an individual to provide protectionagainst or to treat on-going infection caused by the above organisms.From this, it will be appreciated that the immunogenic vaccineconjugates of the invention will be a source of therapeutic antiserum inlight of their favorable immunogenicity with minimal inducement of GBMPcross-reactive antibodies. The conjugates of the invention will also beuseful for raising monoclonal antibodies and, possibly, antidiotypeantibodies.

[0013] We have found that most of the bactericidal and protectiveantibodies induced by the N-Pr-GBMP-protein conjugate described in theabove-referred to Jennings et al U.S. Pat. No. 4,727,136 are notassociated with the GBMP cross-reactive antibodies. In fact, most of theprotective activity is contained in an N-Pr-GBMP-specific antibodypopulation which does not cross-react with GBMP. In light of this, it isbelieved that the N-Pr-GBMP mimics a unique bactericidal epitope on thesurface of group B meningococci.

[0014] The present invention is based on the discovery that it ispossible to synthesize chemically modified GBMP's which mimic thebactericidal epitope and which, in their conjugated form, not onlyexhibit enhanced immunogenicity but also avoid substantially theinducement of antibodies that do cross-react with GBMP.

[0015] In arriving at the present invention, different chemicallymodified GBMP's have been synthesized and conjugated individually toprotein, followed by injection of the conjugates into mice and theeffects compared to those produced by the N-Pr-GBMP protein conjugate.Surprisingly, it has now been found that the presence of an unsaturatedbond in the N-acyl results in particularly immunogenic conjugates

[0016] These and other features of the invention will be betterunderstood through a study of the following detailed description of aspecific embodiment of the invention. The scope of the invention islimited only through the claims appended hereto.

DETAILED DESCRIPTION OF THE INVENTION

[0017] This invention generally provides novel group B Neisseriameningitidis unsaturated N-acyl derivative polysaccharides, novelconjugates of the group B unsaturated N-acyl derivatives, pharmaceuticalcompositions comprising conjugate molecules of group B Neisseriameningitidis unsaturated N-acyl derivative polysaccharide fragmentscovalently bound to proteins, and the use of these compositions asvaccines.

[0018] The present invention relates to group B N. meningitidisunsaturated N-acyl derivative polysaccharides of Formula (I):

[0019] wherein R₁ is a C₂-C₄ unsaturated alkyl group comprising at leastone double bond.

[0020] In one embodiment of the invention, R₁ of Formula I has three, orfour carbons and two nonadjacent double bonds.

[0021] In a further embodiment of the invention, R₁ of Formula I is two,three, or four carbons, and the carbon most distant from the acyl carbonis bound through a double bond.

[0022] Specific, but not limiting examples of modified group Bmeningococcal polysaecharide N-acyl derivative polysaecharides ofFormula I useful in the present invention include the following

[0023] The group B meningococcal polysaccharide is isolated from N.meningitidis by methods which are known in the art. In one such method,group B meningococci (strain 981B) were grown at 37° C. in a fermenterusing 30 g. of dehydrated Todd Hewitt Broth (Difco Laboratories,Detroit, Mich.) per liter of distilled water. Prior to fermenter growth,the lyophilized strain was grown initially in a candle jar at 37° C. on5% (v/v) Sheeps' Blood Agar (Difco Laboratories, Detroit, Mich.) plates.The bacteria were then transferred to 1.0 liter of Todd Hewitt Broth (asabove) in an Erlemneyer flask which was shaken at 37° C. for 7 hours at190 r.p.m. This inoculum was then transferred to the fermenter. Afterfermenter growth (16 hours) the bacteria were killed by the addition offormalin to a final concentration of 0.75%. The bacteria were removed bycontinuous centrifugation and the group B meningococcal polysaccharidewas isolated from the supernatant and purified essentially as describedby Bundle et al, J. Biol. Chem., 249, 4797-4801 (1974) except that theprotein was extracted by stirring a solution of the crude polysaccharidewith cold (4° C.) 90% phenol instead of hot (50-60° C.). This latterprocess ensures that a high molecular weight form of the GBMP isproduced.

[0024]E. coli (018:K1:H7) (NRCC 4283) were grown at 37° C. in afermenter in distilled water containing dehydrated Brain Heart Infusion(BHI; 37 g/litre) (Difco Laboratories, Detroit, Mich.). Prior tofermenter growth, the lyophilized strain was grown on 50 ml of BHIsolution (same as above) in an Erlenmeyer flask which was shaken at 37°C. for 7 hours at 200 r.p.m. This growth was then transferred to 1.5litres of BHI (as above) and grown under the same conditions asdescribed above for 7 hours. The inoculum was then transferred to thefermenter.

[0025] The procedures employed in the isolation and purification of thecapsular polysaccharide of E. coli K1 were identical to those describedabove for the isolation of the group B meningococcal polysaccharide.

[0026] It will be appreciated that the isolation and purificationprocedures described above are not the only ones which may be utilized,and that other published procedures are available, for example thosedescribed by Watson et al, J. Immunol. 81, 331 (1958) and in theabove-mentioned U.S. Pat. No. 4,727,136.

[0027] The native polysaccharide is N-deacetylated to provide a reactiveamine group in the sialic acid residue parts of the molecule. TheN-deacetylation can be carried out by any known method, for example in abasic aqueous medium at elevated temperatures, for example about 90° to110° C., and at a pH of about 13 to 14. The basic aqueous medium issuitably an aqueous alkli metal hydroxide solution, for example sodiumhydroxide of about 2M concentration. Alternatively, hydrazine in aqueoussolution may be used. The degree of N-deacetylation may vary from about30% to 100% depending on the conditions. It is preferred to achieveabout 90 to 100% N-deacetylation. N-deacetylated product can berecovered for example by cooling, neutralizing, purification if desired,and lyophilization.

[0028] As a result of N-deacetylation, fragments of the polysaccharideare usually produced having an average molecular weight ranging fromabout 3,000 to 50,000 Daltons. For use in this invention, fragments orfull length polysaccharides may be used.

[0029] The N-deacetylated polysaccharide fragments or full lengthpolysaccharides are then N-acylated to produce the correspondingN-acylated product. The N-acylation may be carried out by dissolving theN-deacetylated polysaccharide in an aqueous buffered medium having a pHof about 7.5 to 9.0, followed by adding the appropriate unsaturated acylanhydride, optionally with an alcohol to increase solubility, andcooling to below 10° C. until the reaction is complete. If desired, thereaction medium can be purified. Non-limiting examples of purificationmethods which may be utilized include dialysis followed by recovery ofthe N-acylated product by lyophilization. The reaction is substantiallycomplete within about 10 to 20 hours. The degree of N-acylation, asmeasured by analytical techniques, typically ¹H nmr, is at least 90% andmore likely close to 100%. The N-acylation reaction does not result inany significant molecular weight reduction of the fragments.

[0030] The conjugate molecules of this invention have the formula II

[0031] wherein R₂ is an unsaturated C₂₋₄ acyl group. The conjugatestherefore may comprise the unsaturated polysaccharides of this inventionand may also include the acryloyl derivative.

[0032] It is preferred, according to the present invention, to selectfor conjugation purposes the C₂₋₄ N-acylated material having an averagemolecular weight corresponding to about 10 to 200 sialic acid residues.Thus, a preferred conjugate is the N-acryloyl (2-propeneoyl) derivative.This is generally achieved by way of gel filtration of the N-acylatedGBMP using an Ultragel trademark AcA 44 (Bead diameter 60-140 um)column, using PBS as eluant. Alternatively, a suitable sizing membranemay be employed.

[0033] Unsaturated N-acylated material of average molecular weight of30,000 to 40,000 Daltons, for example 10,000 to 15,000 Daltons, ispreferably employed for the invention. This is obtained by collectingthe fractions of the eluate of the column containing N-acylated GBMPmaterial having that average molecular weight range. N-acylated materialof higher average molecular weight, for example in the region of 30,000to 40,000 Daltons, has also proved to be useful according to theinvention.

[0034] The molar ratio of polysaccharide to protein in the conjugatemolecules of the invention is preferably between 1 mole protein to 20moles polysaccharide. More preferably the ratio is between 1 moleprotein and about 2-15 mole polysaccharide. Most preferably the ratio isabout 1 mole protein and 4 to 7 moles polysaccharide. Variations inprotein/polysaccharide ratio may be achieved by adjusting the ratio ofthe starting components in the conjugation reaction.

[0035] In addition to providing conjugate molecules comprisingunsaturated N-acyl derivative polysaccharides conjugated to protein,this invention also contemplates multivalent conjugates and theirvaccines wherein different types of polysaccharides are conjugated to asingle protein.

[0036] The vaccines of the invention are produced by conjugating theunsaturated N-acylated polysaccharide with an immunologically suitablecarrier protein. Preferably, the carrier protein itself is an immunogen.Non-limiting examples of suitable carrier proteins are bacterialproteins, or polypeptides including tetanus toxoid, diphtheria toxoid,cross-reacting materials (CRMs), preferably CRM_(197′) (obtained fromSclavo Ltd., Siena, Italy), and bacterial protein carriers, such asmeningococcal outer membrane proteins.

[0037] Any mode of conjugation may be employed to conjugate the modifiedpolysaccharide fragments with the carrier protein. A preferred method isthat described in U.S. Pat. No. 4,356,170, i.e. by introducing terminalaldehyde groups (via oxidation of cis-vicinal hydroxyl groups) into theN-acylated polysaccharide and coupling the aldehyde groups to theprotein amino groups by reductive amination. The polysaccharide and theprotein are thereby linked through a —CH₂—NH—protein linkage.

[0038] It is to be understood, however, that the conjugate vaccines ofthe invention are not limited to those produced via reductive amination.Thus, the vaccines may also be produced by conjugating the N-acylatedpolysaccharide with the carrier protein using an adipic dihydrazidespacer, as described by Schneerson, R., et al, Preparation,Characterization and Immunogenicity of Haemophilus influenzae type bPolysaccharide-Protein Conjugates, J. Exp. Med., 1952, 361-476 (1980),and in U.S. Pat. No. 4,644,059 to Lance K. Gordon. Alternatively, thebinary spacer technology developed by Merck may be used, as described byMatburg, S., et al, “Biomolecular Chemistry of Macromolecules: Synthesisof Bacterial Polysaccharide Conjugates with Neisseria meningitidisMembrane Protein”, J. Am. Chem. Soc., 108, 5282-5287 (1986) or,possibly, the reducing ends methodology.

[0039] The conjugate molecules prepared according to this inventiontypically comprise a protein to which is bound at least onemeningococcal polysaccharide fragment of the present invention through asingle binding site at the terminal end of the backbone of thepolysaccharide fragment. Thus, this invention provides the ability, ifdesired, to produce, meningococcal conjugate molecules wherein thepolysaccharide component, except for one end, is unobscured by protein.Other methods of conjugating meningococcal polysaccharides to proteinthrough the terminal sialic acids of the branches may, result incrosslinking, and attachment of polysaccharide to protein at a pluralityof sites. This invention also contemplates conjugate molecules which maybe made using a combination of methods.

[0040] The resulting N-acylated polysaccharide protein conjugates whichdo not possess significant cross-linking are soluble in aqueoussolutions. This makes these conjugates of the invention particularlygood candidates for vaccine use.

[0041] A resulting unsaturated N-acylated-polysaccharide proteinconjugate of the invention has been tested in in vitro tests in mice,and has been shown to possess improved immunogenic properties ascompared with the N-propionylated-polysaccharide. In addition,substantially reduced formation of cross-reactive antibodies isobserved. In addition, the unsaturated conjugate demonstrated unexpectedhigh bactericidal titers compared to other conjugates tested. In lightof this, it is believed that the vaccines of the invention will beuseful against meningitis caused by group B N. meningitidis or by E.coli K1 organisms. Of particular interest are vaccines for protectinghuman infants who are most susceptible to bacterial meningitis.

[0042] The vaccines of this invention may comprise standard carriers,buffers or preservatives known to those in the art which are suitablefor vaccines. In addition, adjuvants such as alum or stearyl tyrosinemay also be included in the formulation to enhance the immunogenicresponse.

[0043] The vaccines of the present invention are typically formed bydispersing the conjugate in any suitable pharmaceutically acceptablecarrier, such as physiological saline or other injectable liquids. Theadministration of the vaccine of the present invention may be effectedby any of the well-known methods, including, but not limited tosubcutaneously, intraperitonealy or intramuscularly. The preferredmethod of administration of the vaccine is parenteral administration.Additives customary in vaccines may also be present, for examplestabilizers such as lactose or sorbitol and adjuvants such as aluminumphosphate, hydroxide, or sulphate.

[0044] The vaccines of the present invention are administered in amountssufficient to provoke an immunogenic response. Typically a dose ofbetween about 1 and 50 gg polysaccharide is effective for generatingsuch a response. Dosages may be adjusted based on the size, weight orage of the individual receiving the vaccine. The antibody response in anindividual can be monitored by assaying for antibody titer orbactericidal activity and boosted if necessary to enhance the response.

[0045] A suitable dosage for the vaccine for human infants is generallywithin the range of about 5 to 25 micrograms, or about 1 to 10micrograms per kilogram of body weight.

EXAMPLES

[0046] The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way.

Example 1 Synthesis of N-Acryloylated GBMP

[0047] Synthesis of N-acryloylated GBMP is described in Roy R. et al.,Glycoconjugate J. (1990) 7:3-12. N-Deacteylated GBMP (150 mg) wasdissolved in 2.0 ml of distilled water. The solution was cooled to 0° C.and treated with 50 μl (1 eq) installments of acryloyl chloride (AldrichChemical Co.) for a totals of 500 μl. The pH of the solution wasmaintained at pH 8.5 with 4M NaOH using an autotitration unit. After thecomplete addition of the acid chloride (2 hr.), the pH was raised to 12and maintained at this level for 30 minutes. The material was purifiedby exhaustive dialysis against distilled water at 4° C., followed bylyophilization to 163 mg. H-NMR of the material revealed 100%N-acylation with the appropriate integration pattern for the acryloylsubstituent.

Example 2 Activation of N-Acryloylated GBMP

[0048] N-acryloylated GBMP (150 mg) was dissolved in distilled water(1.25 ml) followed by the addition of 3.75 ml of a 0.2M solution (˜50eq) of NaIO₄ in water. The solution was kept in the dark at roomtemperature for 1 hour, followed by the addition of ethylene glycol (400μl, 10 eq).After 1 hour at room temperature, the solution was applieddirectly to a Sephade G-10 (1.6×100) column equilibrated in water(Pharmacia Fine Chemicals). The activated product was eluted off thecolumn in the void volume peak, which was collected and lyophilized. Theoxidized product i was then fractionated on a BioGel A.5 column(1.6×100) (BioRad) equilibrated in phosphate buffered saline (pH 7.6).Molecular weight pools were made bases on HPLC (high performance liquidchromatography) analysis (Pharmacia-Superose, 12 column) of selectedfractions of the eluted material. Comparison of the relative Kav valueof each fraction to a previously constructed calibration curve allowedfor the selection of a discrete 11 KD fractions of oxidized acryloylatedGBMP. The factions were purified by dialysis as described above. H-NMRspectroscopy of the fractionated material was consistent with oxidizedN-acrylolyated GBMP.

Example 3 Preparation of a Tetanus Toxoid Conjugate of N-AcryloylatedGBMP

[0049] Freshly purified tetanus toxoid monomer (TT-m; 3.5 mg) wascombined in a Pierce reacti-vial with 10.5 mg of an 11 KD fraction ofoxidized acryloylated GBMP. Sodium cyanoborohydride (7.0 mg) was addedand the mixture was dissolved in 233 μl of phosphate buffer (0.1 M, pH7.5). The solution was incubated at 37° C. for a total of five days.Periodically, the conjugation was monitored by size exclusion HPLC(Superose-12, Pharmacia) to visualize the shift to higher molecularweight as the conjugation progressed. The final conjugate was purifiedfrom starting materials by fractionation over a BioGel A.5 columnequilibrated in PBS, followed by dialysis, and lyophilization.Colorimetric analysis for total sialic acid (Svennerholm method) andprotein (BCA method, Pierce) indicated conjugates that contained between12-30% sialic acid.

Example 4 Immunization of Mice

[0050] Typically, 10 female CF1 mice (8-10 weeks old) were immunizedintraperitoneally (0.2 ml) with an amount of conjugate equivalent to 2μg of sialic acid, with or without the addition of adjuvants such asAlum (Alhydrogel₉ Superfos Biosector) or RIBI's complete or componentadjuvant system (RIBI Immunochem). The initial vaccination was followedby booster vaccinations on day 21 and day 35, followed by exsanguinationon day 45. The blood was collected via heart puncture and the serumstored aliquoted at −86° C.

Example 5 Bactericidal Assay

[0051] The bactericidal assay was carried out in tissue culture 96 wellmicrotiter plates (Coming). All antisera were heat inactivated at 56° C.for 30 minutes prior to their use. Group B meningococcus (strain 80-165B;2b:p.1) was grown overnight on chocolate agar plates (QueLab) at 37°C. under a 5% CO₂ atmosphere, followed by inoculating a second plate andincubating it for five hours. The appropriate dilutions of antisera weremade directly in the plate using Hank's balanced salt solution (HBSS) asthe diluent to yield a final volume of 50 μl per well. A suspension ofGBM in HBSS was made giving an O.D. (λ₅₈₀)=0.1 Absorbance. Thissuspension was diluted 40,000 times in HBSS to give the final workingdilution of bacteria for the assay. Freshly thawed baby rabbitcomplement (Pel-Preeze Biologicals) was added (20 μl) to each well,followed by 30 μl of the working dilution of bacteria. The plate wasthen shaken at 37° C. for one hour. The contents of each well was mixedprior to plating (35 μl ) onto chocolate agar plates. The plates werethen incubated overnight at 37° C./5% CO₂ and the number of colonyforming units (CFU) were counted. The % killing was determined relativeto the mean values of either HBSS control wells or an irrelevantantiserum in the following manner:

% killing=(CFU _(control) −CFU _(antiserum) /CFU _(control))×100

Example 6

[0052] Passive Protection Assay

[0053] Mouse antisera obtained from the N-Acyl GBMP-TT immunizationswere typically diluted in sterile saline or PBS (phosphate bufferedsaline). Groups of five female CF1 mice (8-10) weeks old were injectedintravenously with 200 μl of the diluted antisera. After one hour, eachgroup of mice was challenged with an intraperitoneal injection (500 μl;800-1200 CFU/ml) of a suspension of Group B Neisseria meningitidis (GMB80165 B:2b:P.1). After five hours, the blood was harvested from for theindividual mice by cardiac puncture and 10 μl of the blood was platedonto chocolate agar plates. The plates were incubated at 37° C. under 5%CO₂ and the number of colony forming units (CFU's) were determined 15-20hours later.

[0054] The passive protection assay is based on the reduction orclearance of bacteria in the presence of specific antibody and ismeasured relative to a control group lacking specific antibody. Thedegree of protection offered by the mouse anti-N-Acyl GBMP conjugatesera is represented by the % reduction of CFU's for each antiserumrelative to an irrelevant control antiserum or PBS.

Example 7 Synthesis and Biological Activity of a Vaccine AgainstNeisseria meningitidis serogroup B

[0055] The new conjugate vaccine against N. meningitidis serogroup B wassynthesized, the design of which is based on a unique modification ofthe native polysaccharide. The native polysaccharide (N—Ac GBMP) wasderivatized at the amino terminus by complete substitution of theN-acetyl groups with N-acryloyl groups (NH—CO—CH═CH₂). Physical methods,such as ¹H and ¹³C-NMR spectroscopy characterized with certainty theidentity and homogeneity of the new species and size exclusion HPLCdemonstrated that the process did not alter the molecular size of thepolysaccharide through depolymerization. Conjugates to proteins weremade in a manner to similarly described procedures. Briefly, startingfrom preparation of the N-acryloyl GBM polysaccharide, two differentlots of N-acryloyl GBMP-tetanus toxoid conjugates were prepared.Colorimetric analysis of each conjugate revealed a 13% and 20% totalsialic acid to conjugate ratio, respectively. ¹H-NMR spectroscopy of theconjugates revealed the unchanged presence of the modifiedpolysaccharide on the protein.

[0056] In separate animal experiments, the N-Acrolyl GBMP-TT conjugateswere injected into mice in conjunction with either saline, aluminumhydroxide, or RIBI's complete adjuvant (MPL+TDM+CWS) in one instance,and with RIBI's adjuvant only in the second case. The vaccines werevisibly well tolerated in mice.

[0057] Serological testing of each antiserum showed that both conjugateselicited a specific response comparable or higher than those seen withN-propionyl GBMP-TT constructs using the RIBI's adjuvant system (SeeTable 1). Preliminary studies regarding the cross reactivity of theN-acryloyl GBMP-TT antisera showed results that were similar to thedegree of cross reactivity seen with N-propionyl GBMP-TT antisera (SeeTable 2). One of the two lots of N-acryloyl GBMP-TT antisera showedsignificantly less cross reaction to the native GMBP relative to anN-propionyl GBMP-TT construct administered in the same experiment.

[0058] Both lots of N-acryloyl GBMP-TT were tested for theirbactericidal activity against live GBM and have shown significantactivity relative to N-propionyl GBMP-TT antisera. These results aresummarized in Tables 1 and 3. The results in Table 1 are the product ofa bactericidal assay performed in duplicate and are consistent withdilution values found with other assays performed with the samematerial. The data of Table 1 are consistent with acryloyl possessingparticularly effective bactericidal activity. Table 3 compares thebactericidal activity of the two lots of N-acryloyl GBMP-TT antiseratogether with N-propionyl GBMP-TT antisera obtained in the same animalexperiments. The assay uses a 15-fold greater number of bacteria andhence only those antisera showing strong activity were detected. From acomparison of the N-acryloyl GBMP-TT antisera to N-propionyl GBMP-TTantisera, it can be seen that the bactericidal activities are virtuallyequivalent.

[0059] Passive protection studies were carried out with varyingdilutions, all of which demonstrated significant clearance, thusinferring protection to the mice. (See Table 1). Comparison to theN-propionyl GBMP-TT antiserum at the different dilutions gave againnearly identical results. Comparison of the two lots of N-acryloylGBMP-TT antisera in a passive protection experiment showed that bothprotected the mice identically, within experimental error. (See Table3). TABLE 1 Summary of experiment RPV-1-63 comparing the properties ofmodified group B meningococcal polysaccharide-tetanus toxoid conjugatesin various adjuvants Bactericidal ELISA Titer^(b) Passive ProtectionTiter^(c) Titer^(a) 90% 50% % Clearance % Clearance % ClearanceAntiserum Saline Alum RIBI Killing Killing (neat) (1:4) (1:6)N-Propionyl GBMP-TT 14,387 38,667 235,456 210 690 100 81 73 N-ButanoylGBMP-TT 11,253 38,859 363,264 9.2 50 60 — 0 N-Penatanoyl GBMP-TT 14,01953,248 465,920 12.1 28 57 — 0 N-Acryloyl GBMP-TT 1,698 7,280 218,3041000 2,120 96 78 46

[0060] TABLE 2 Cross reaction of the modified N-acyl GBMP-TT antisera(RPV-1-63) to native N-acetyl GBMP antigen ELISA Titer^(a) Saline AlumRIBI N-Acyl^(b) N-Acyl^(c) Ratio of N-Acyl/ N-Acyl N-Acetyl Ratio ofN-Acyl/ N-Acyl N-Acetyl Ratio of N-Acyl/ Antiserum titer titer N-Acetyltiter titer titer N-Acetyl titer titer titer N-Acetyl titer N-PropionylGBMP-TT 2,557 53 49 6,217 795 8 51,373 2,910 18 N-Butanoyl GBMP-TT 2,62845 59 10,979 226 49 66,267 406 163 N-Penatanoyl GBMP-TT 2,764 9 3146,491 150 43 120,533 311 388 N-Acryloyl GBMP-TT 338 7 46 3,022 546 650,040 1,100 45

[0061] TABLE 3 Summary of the protective properties from two lots ofN-Acrylovl GBMP-TT antisera relative to N-Propionvl GBMP-TT antiseraBactericidal activity^(b) Passive protection^(c) Antiserum^(a) 50%killing % clearance N-Propionyl GBMP-TT 53 92 (RPV-1-45) N-AcryloylGBMP-TT 80 100 (RPV-1-45) N-Propionyl GBMP-TT 29 81 (RPV-1-63)N-Acryloyl GBMP-TT 100 78 (RPV-1-63)

Example 8 Further Studies Using N-Acyl Modified GBMP-TT Conjugates

[0062] A series of N-acyl modified GBM polysaccharides (N-propionyl GBMP(NPr), N-butanoyl GBMP (NBu), N.-pentanoyl GBMP (NPe), and N-acryloylGBMP (NAcryl) were synthesized essentially as previously described withthe exception of using pH control to limit depolymerization of thepolysaccharides. ¹H- and ¹³C-NMR spectroscopy allowed completeidentification of the modified polysaccharides, and it was determinedthat each polysaccharide was 100% derivatized. A series of oxidizedpolysaccharide fragments of the same molecular weight (11 KD) weregenerated based on SEC-HPLC profiled run on a standardized column. Allof the conjugates were synthesized under the exact same conditions.Colorimetric analysis of the conjugates yielded the following sialicacid incorporation: NPr-28%, NBu-30%, NPe-18%, NAcryl-19%.

[0063] Mice were immunized with 2 μg of sialic acid/conjugate either insaline, absorbed onto aluminum hydroxide, or emulsified in RIBI'sadjuvant. All of the conjugates were well tolerated in the mice with novisible signs of malaise.

[0064] ELISA titrations of the various antisera against homologouspolysaccharide antigens are summarized in Table 1. The adjuvantproducing the highest titers was found to be the RIBI's series increasesfrom N-propionyl to N-pentanoyl substantiating previous findings usingother hydrophobic adjuvant systems. In an adjuvant system such as alum,there does not appear to be a corresponding trend in titer. Specificitytowards the immunizing polysaccharide also increases with increasinglength of the acyl chain from N-propionyl to N-pentanoyl, most markedlyin the RIBI series. (See Table 2). This result is also in accord withprevious results which demonstrated the same trend. Despite the increasein titer and specificity, there is not an associated increase inactivity against the native bacteria both in bactericidal and passiveproduction assays. The N—Pr antiserum shows significantly higherbactericidal titers (14-25 times higher) at the 50 and 90% levelsrelative to the N—Bu and N—Pe antisera. Correspondingly, passiveprotection studies at different dilutions of antisera show significantclearance of the bacteria with the N-Bu and N-Pe at only the highestconcentration, unlike the N-Pr antiserum which shows significantclearing even at 1:6 dilutions.

What is claimed is:
 1. A modified group B meningococcal polysaccharidewherein the N-acetyl groups are substituted with an N-acyl derivative asin Formula (I):

wherein R₁ is a C₃-C₄ unsaturated alkyl group comprising at least onedouble bond.
 2. The polysaccharide of claim 1 wherein R₁ is four carbonsand has two non-adjacent double bonds.
 3. The polysaccharide of claim 1wherein R₁ is four carbons and has one double bond.
 4. Thepolysaccharide of claim 1 wherein R₁ is three carbons.
 5. Thepolysaccharide of claim 2 wherein R₁ is CH₂═CH—CH₂—CH₂.
 6. Thepolysaccharide of claim 3 wherein R₁ is CH₂═CH—CH₂.
 7. Thepolysaccharide of claim 1 wherein the carbon most distant from the acylcarbon is bound through a double bond.
 8. The polysaccharide of claim 7wherein R₁ is four carbons.
 9. The polysaccharide of claim 7 wherein R₁is three carbons.
 10. A conjugate molecule comprising at least onepolysaccharide fragment of Formula (II),

wherein R₂ is an unsaturated acyl group comprising at least one doublebond and the fragment is covalently bound to a protein.
 11. Theconjugate molecule according to claim 10 wherein the protein is derivedfrom a bacteria.
 12. The conjugate molecule according to claim 11wherein the bacteria is Neisseria meningitidis.
 13. The conjugatemolecule according to claim 10 wherein the protein is derived from abacteria selected from the group consisting of tetanus toxoid,diphtheria toxoid, CRM₁₉₇, and meningococcal outer membrane proteins(OMP).
 14. The conjugate molecule according to claim 10 wherein thepolysaccharide fragment is an N-acyl derivative polysaccharide, theprotein is tetanus toxoid and DMP and the molecular weight of thepolysaccharide fragment is between about 3 kDa and 50 kDa.
 15. Theconjugate molecule according to claim 10 wherein the polysaccharidefragment is an N-acyl derivative polysaccharide, the protein is tetanustoxoid and the molecular weight of the polysaccharide fragment isbetween about 10,000.
 16. The conjugate molecule according to claim 19wherein the molar ratio of polysaccharide to protein is between about 20polysaccharide and 1 protein.
 17. The conjugate molecules according toclaim 20 wherein the molar ratio of polysaccharide to protein is about 4to 7 moles of polysaccharide to about 1 mole protein.
 18. The conjugatemolecule according to claim 10 comprising N-acyl derivative fragments.19. A vaccine composition of conjugate molecules comprising a group Bmeningococcal unsaturated C₃₋₅ N-acyl derivative polysaccharide fragmentcovalently bound to a protein.
 20. The vaccine composition according toclaim 19 wherein the protein component is derived from a bacteriaselected from the group consisting of tetanus toxoid, diphtheria toxoid,CRM₁₉₇, and meningococcal outer membrane proteins.
 21. The vaccinecomposition according to claim 19 wherein the fragment is an N-acylderivative polysaccharide and the molecular weight of the polysaccharidefragment is between about 3 and 50 kDa.
 22. The vaccine compositionaccording to claim 19 wherein the protein component is derived fromNeisseria meningitidis.
 23. An immune serum comprising antibodies raisedin a mammal immunized with the conjugate according to claim
 10. 24. Theimmune serum according to claim 23 wherein the polysaccharides of theconjugate are fragments of unsaturated C₃₋₅ N-acyl derivativepolysaccharides.
 25. A method of immunizing a mammal against Neisseriameningitidis and E. coli K1 infections comprising administering to themammal an immunizing amount of the vaccine according to claim 19.